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Abstract:
The complexity of development, growth and disease in model organisms such as the mouse offer a major challenge to new micro-imaging technologies that have been developed over the last decade. Each modality has its strengths and weaknesses for applications in molecular and preclinical imaging. Recently, ultrasound imaging technology has been scaled in terms of frequency to provide much higher resolution over smaller fields of view making it suitable for animal model systems imaging. This approach, referred to as ultrasound biomicroscopy (UBM), provides realtime (30 Hz) high resolution (30 - 100 micrometers) structural imaging over a depth of approximately 15 mm. In addition, functional data on blood flow in the vessels is provided by a targeted (duplex) Doppler. In this presentation, the potential of ultrasound micro-imaging to contribute to the visualization and analysis of model systems is reviewed. The development of high frequency imaging instrumentation will be described and specific examples of applications in developmental biology (chick embryo, xenopus), cardiovascular development in the mouse, and cancer models in the mouse will be presented. Technological innovations such as ECG gated 1000 Hz imaging of the mouse heart, and ultrasound guided microinjection will be shown. Finally, aspects of targeted, microbubble and nanoparticle contrast agents relevant to molecular imaging will be discussed.

Abstract: [ Download abstract ]
A major challenge for diagnostic imaging is to better define the specific characteristics of individual tumors. Tumors sharing a particular histopathologic type may have widely divergent biological properties including molecular expression, angiogenesis status, and susceptibility to treatment. Through the use of innovative contrast enhancing pharmaceuticals, it now seems feasible to interrogate each patientbd structural characteristics. One of the several appealing approaches now being developed for clinical use is the use of dynamic magnetic resonance imaging combined with novel macromolecular contrast media to quantitatively assess tumor microvessels. Using computer processing of the imaging data and a relatively simple two-compartment kinetic model, it is possible to non-invasively assay the relative blood volume, microvascular endothelial leakiness, and the interstitial volume of any solid tumor. The MRI permeability assay appears to be a particularly powerful biomarker, able to indicate a tumorbogenic status, and the tumorb drug therapy. Using MRI and macromolecular contrast media, tumor responses to treatment can be detected within hours of initiating therapy. Optimization of the dynamic MRI technique linked with the anticipated availability of new macromolecular contrast media should lead to an even greater future benefit from diagnostic imaging in cancer patients.

Learning objectives:

To comprehend the diagnostic benefits afforded by MRI enhanced with novel macromolecular contrast agents for the characterization of individual tumors.

To understand the principles of altered microvascular permeability and how abnormal permeability relates to angiogeneisis and tumor aggressiveness.

To learn how the effects of anti-angiogenic therapy can be monitored by macromolecular enhanced MRI.

Abstract: [ Download abstract ]
Molecular imaging and medicine can benefit from the discovery of new tissue-specific targets that are inherently accessible to agents injected intravenously. Using our recently developed tissue subfractionation techniques for purifying luminal endothelial cell plasma membranes and caveolae directly from normal organs as well as tumors, we have generated multiple distinct high-resolution proteomic maps of the endothelial cell surface directly in contact with the circulation. We have used mass spectrometry, de novo sequencing, and database searching coupled with antibody generation for immunoblotting, tissue immunostaining, intravital microscopy, and SPECT imaging in vivo to compile an extensive proteomic database including multiple validated IV-accessible vascular targets specific to single organs or solid tumors. Novel organ- and even tumor-induced targets have been identified in mouse, rat, monkey and human tissues and then used to image the vasculature of whole organs and tumors selectively after intravenous injection of monoclonal antibodies in rodents. SPECT imaging rapidly visualizes the tissue-specificity of immunotargeting with high sensitivity and objectivity. Our new antibodies show clearly that tissue-specific delivery to individual solid tumors or single normal organs can indeed be achieved in vivo at levels approaching the theoretical expectations of the vascular targeting strategy. Up to 90% of the IV-injected dose of antibody can accumulate in a single tissue within 30 min. For antibodies specifically targeting caveolae, both tissue staining and live dynamic imaging by intravital microscopy reveal rapid targeting of a single vascular bed with extensive transport across the endothelial cell barrier leading to much improved tissue penetration. Electron microscopy shows additional details including selective antibody entry and binding within caveolae followed by rapid transcytosis and uptake by underlying cells inside the tissue. Our novel profiling strategy directly reveals distinct molecular signatures for the endothelial cell surface in each major organ (lung, kidney, liver, heart & brain) and in solid tumors including breast, lung, kidney, liver, brain and prostate. This new development and optimization of several key technologies encompassing tissue subfractionation, subtractive proteomics and molecular imaging permits rapid discovery and validation of tissue-specific targets that are inherently accessible to agents injected intravenously. Profiling IV-accessible vascular & caveolae targets is an important logical step not only for achieving site-directed pharmacodelivery & selective molecular imaging in vivo but also for overcoming the normally restrictive endothelial cell barrier to transport drugs & even genes to their intended target cells inside the tissue.

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Gene transfer strategies have been developed to re-direct the antigen specificity of T-cells by expressing chimeric immunoreceptors that engage cell-surface epitopes on tumor cells. FDA-authorized clinical trials are proceeding with early feasibility and safety studies in humans. The City of Hope's Cancer Immunotherapeutics Program has four IND's active exploring the feasibility and safety of adoptive T-cell therapy using genetically modified chimeric immunoreceptor re-directed cytolytic T-cell clones for lymphoma, neuroblastoma, and glioblastoma. Engineered T-cells in these protocols co-express the HyTK fusion protein thus providing the opportunity to image these T-cells in humans using HSV-TK molecular PET probes such as 18FHBG and FIAU. Under consideration is the first human studies to image therapeutic HyTK+ T-cells with 18FHBG in the setting of intracranial T-cell adoptive therapy for glioblastoma multiforme.

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Fluorescence imaging is a powerful modality that is increasingly used for gene-expression profiling, probing protein function and elucidating cellular pathways. Fluorescence generated in in-vitro assays can be easily quantified using fluorometers or charge coupled devices (CCD). Similarly, fluorescence of superficial structures has been imaged in vivo usingintravital, confocal or multiphoton microscopy. Quantitation and imaging of fluorescence in deeper tissues however has been more elusive. This talk describes current progress with instruments and methods for in-vivo imaging and tomography of whole animals using Fluorescence MolecularTomography (FMT). We show the capacity to resolve fluorescent objects embedded deep in mouse-like phantoms achieving sub-millimeter resolution. We further demonstrate how quantification and high molecular specificity can be achieved and that penetration depths of several centimetres are feasible. Examples of imaging enzyme up-regulation, induced apoptosis and gene-expression in-vivo are given. Limitations of the method and future directions are also discussed.

A Tale of Two Tracers: New Myocardial Perfusion Agents and Monitoring Gene Therapy for Parkinson's Desease.

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Coronary artery disease (CAD) is the greatest cause of morbidity and mortality in the USA and the greatest health care cost. Currently, myocardial perfusion scintigraphy (MPS) is the most widely applied noninvasive imaging method for the diagnosis, localization and risk stratification of patients with known or suspected CAD. New SPECT and PET probes have been developed based on the natural product rotenone that exhibit myocardial extraction and retention properties superior to the that of the current clinical agents, 99mTc-sestamibi and thallium-201. A variety of imaging modalities are being applied to drug development. For gene therapy several key questions, such as is the gene is being delivered to the appropriate tissue and is the gene being expressed, can be answered using imaging techniques. One application is the development of gene therapy for Parkinson's Disease where replacement of the DOPA decarboxylase gene can be monitored by the PET reporter probe [18F]fluoro-meta-tyrosine. Results from the ongoing development of this therapy/ imaging fusion are promising and phase I human trials incorporating imaging are planned.

Luciferase Genes as Reporters of Genes Expression: Diversity and Biochemistry.

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Genes for luciferases, which catalyze bioluminescent reactions, are now widely used as reporters for gene expression in a diversity of applications. This lecture will review the many different luciferases, their genes, biochemistries and applications.

Bioluminescence derives from a luciferase-catalyzed chemiluminescence, a highly exergonic reaction involving oxidation of a luciferin substrate to give a peroxy intermediate. The energy released is used to produce an intermediate or product in an electronically excited state, which then emits a photon. It does not come from or depend on light absorbed, as in fluorescence or phosphorescence, but excited states produced in such reactions are indistinguishable from those produced in fluorescence. In some systems, there is a secondary emitter, such as GFP in coelenterates.

There are numerous (~ 30) different extant bioluminescent systems, which for the most part bear no evolutionary relationships with one another. The many different luciferases are thus considered to have arisen de novo and evolved independently, and the luciferins are likewise different. Four of these systems, bacteria, dinoflagellates, beetles and coelenterates, will be considered in detail.

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With the wealth of information provided by the maturing areas of genomics and proteomics, the identification of molecular markers and targets now promises contrast-enhanced, diagnostic imaging with sensitivity and specificity that is not possible with conventional, anatomical imaging. While nuclear imaging approaches represent the gold-standard of "molecular imaging" both in tomographic and scintigraphy modes, near-infrared, fluorescence optical imagining has the potential for greater sensitivity and specificity. In addition, fluorescence enhanced optical imaging can be conducted in tomographic and planar imaging modes. In this presentation, the interdisciplinary progress in instrumentation development, chemistry of targeting fluorescent probes, and tomographic imaging algorithms is outlined and placed in the context for small animal imaging for development of molecular therapeutics as well as for clinical, diagnostic imaging.
TBA

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Microwave tomography is a novel imaging modality with attractive biomedical applications. With microwave tomography tissues are differentiated and, consequentially, can be imaged, based on differences in dielectric properties. It has been proven that dielectric properties of biological tissues are a strong indicator of its functional and pathological conditions. This includes tissue blood content, ischemia, infarction, hypoxia, malignancies, edema and others. The method of microwave tomography (as well as electrical field tomography), its applications and imaging results will be discussed.

Abstract:
Tumor hypoxia is an important determinant of relapse-free survival and overall clinical outcome, largely independent of the treatment modality.Â Hypoxic cells are radioresistant and more refractory to certain chemotherapeutic agents. Also, tumor hypoxia is associated with a more aggressive tumor phenotype and hypoxic are more likely to metastasize.

Given the importance of tumor hypoxia in cancer management, much effort is ongoing to develop methods to detect and measure tumor hypoxia.Â One promising approach is to use positron emission tomography (PET) scanning with hypoxia-specific radiotracers, such as 18F-FMISO and 60Cu-ATSM.Â Â AsÂ prelude to clinical studies, we evaluating the compound 124IAZGP, in comparison withÂ 18F-FMISO and 60Cu-ATSM, in laboratory studies.Â Probe measurement of tumor oxygen level is also performed for calibration of the hypoxia images.Â In addition, tumor models with hypoxia-induced transgene expression are used to related the molecular mechanism of tumor hypoxia to the images generated by endogenous markers.

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The technology of modern imaging has advanced to the point that we can detect and measure molecular and biochemical features of cancers (Molecular Imaging). Positron Emission Tomography (PET) is a prime example of an advanced imaging tool that is providing clinical benefit through Molecular Imaging. PET is based on the tracer principle, whereby a biologically important molecule is radiolabeled with a cyclotron produce tracer radionuclide to form a biomedical probe. PET is a sensitive diagnostic technique with very high resolution, and over the last 5 years, PET has been approved for clinical use in important human tumors, including lung, colon, breast, lymphoma, esophageal, thyroid and melanoma. In the last 2 years, PET imaging has been combined with computerized tomography (CT) in the same machine gantry, providing both functional (PET), and anatomic (CT) information in a single fused image. PET/CT is a considered a major technical advance in diagnostic medicine, with the combination device leading to greater diagnostic power than either PET or CT alone. At MSKCC, we have exploited PET/CT applications in clinical research to improve patient care through better diagnosis in thyroid, breast, prostate, colorectal and pediatric cancers. Most of these benefits are based on the single tracer, Fluordeoxyglucose or FDG, a tracer of glycolysis. In the very near future, we anticipate that many different tracers will enter the clinic to allow us to characterize tumors more completely. These tracers will include radiolabeled substrates, drugs, peptides and antibodies. Advances are anticipated in terms of imaging the malignant phenotype;cancer pharmacology, including the pharmacodynamics of targeted therapy; gene expression imaging; imaging cellular events important to cancer, and optimizing radiation therapy through biologic imaging.

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Significant advances have been made over the past five years in developing new technologies for in vivo imaging of small-animal models. This presentation will summarize the latest developments in our laboratory, including applications of the new high-resolution microPET II scanner, hyperspectral approaches to fluorescence tomography, and multimodality PET/CT and PET/MRI imaging. MicroPET II is a dedicated rodent imaging system made up from 17,640 scintillation detectors read out by fiber-optically coupled photomultiplier tubes. In conjunction with maximum a posteriori reconstruction methods, this system routinely achieves ~ 1 mm spatial resolution for in vivo studies at a sensitivity of between 2 and 2.5% depending on the timing and energy window settings. This system is now in use at the UC Davis Center for Molecular and Genomic Imaging, and initial datasets will be shown. We also are developing hyperspectral techniques for 3-D fluorescence imaging in vivo. Using the depth-dependence of the emitted fluorescence light, along with modeling of photon transport in the mouse, promises to offer improvements over intensity-based fluorescence reconstructions. Finally, the integration of anatomical and molecular imaging in the form of PET/CT and PET/MRI imaging systems will be presented and early prototype data from both combinations will be shown.

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In contrast to optical and radioactive emission techniques, magnetic resonance is severely limited in sensitivity. Thus, it is not surprising that in the vast landscape of magnetic resonance phenomena, the term tracer is not found. However, soft tissue contrast, a treasure of contrast mechanisms, and low-impact stable isotopic labels make magnetic resonance molecular imaging an active area of development. Not surprisingly, a significant part of the research is directed toward signal amplification. The dominant water signal is a good source of indirect amplification. Magnetic field strength, and biological amplification at the metabolite level both help. Pre-polarization is a useful strategy, albeit limited by relaxation. This presentation will compare the approaches to using magnetic resonance for molecular imaging, and a take a prospective look at clinical viability. The impact of magnetic field strength, endogenous spectroscopic markers, chemical exchange, carbon-13 labeling, and pre-polarization methods, will all be described. Specific examples include the use of spectroscopy to detect changes in neuronal glutamate levels in MS plaques, the impact of 3T vs 1.5T field strength in prostate MRI/MRSI, amide exchange amplification, and 13C metabolic spectroscopy. The promise of hyperpolarized 13C metabolic imaging will also be discussed.

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We describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer.

Abstract:
Entering the post genomics era the biopharmaceutical industry is faced with an unprecedented number of potential drug targets. Further, advances in gene manipulation and transgene technologies offer the ability to generate mouse models that recapitulates aspects of the clinical disease. As a result, techniques capable of enhancing our understanding of the alter pathophysiology and the influence of therapeutic intervention, at the system level, have become of paramount importance. Biomedical imaging techniques with sufficient resolution and sensitivity for experimental research go some way to meet this need. This presentation will discuss, with specific example, how biomedical imaging impacts preclinical drug discovery and development.